Project Summary The global objective of this research is to elucidate the mechanisms underlying tissue homeostasis and regeneration in mammalian skin and to understand how this process goes awry in human disorders, including cancers. Central to achieving this goal is the characterization of the different stem cells (SCs) within skin, determining their relative contributions to tissue homeostasis and wound-repair, and elucidating how changes in niche microenvironments impact these events. Past AR050452 research led to purification of hair follicle (HF) bulge and basal inter-follicular epidermal (Epd) cells, and established them as long-lived, self-renewing SCs that function in tissue regeneration and wound-repair. However, both in their biology and their tissue regenerative tasks, these SCs display distinct behaviors that appear to be predicated on their unique microenvironments (niches). The field still lacks a comprehensive knowledge of the constituents of these niches, the nature of SC:niche interactions, and how they help stem cells cope with stressful situations. Past AR050452 research has set the foundations to tackle the following key questions: (1). What are the niche components of HFSCs and how do they change with the hair cycle? (2) What are the relative contributions of Epd- and HF- SCs to wound repair? Does this differ in superficial vs deep wounds? In young vs aging mice? (3) What are the changes in SC-niche crosstalk that occur in response to injury and aging and what is their physiological relevance? (4) How do SCs respond to and orchestrate the repair of local breaches in their niche barrier? Which cells transmit damage signals to the surrounding immune cells and how are they triggered to do so? How do SCs survive? (5) How do SCs protect themselves at the end of the hair cycle, when the bulk of the HF undergoes apoptosis and terminal differentiation? To answer these questions, we?ll use FACS, single cell RNA-seq, ChIP- seq, conditional gene knockout and RNAi screens in vivo and employ these methods to explore skin stem cells in their native, mutant and wound-induced environments.